Human papillomaviruses (HPV) are agents responsible for several benign and malignant lesions which proliferate in the skin and mucosal surfaces of humans. They are a genetically diverse group of DNA viruses which infect epithelial tissue, and can cause a range of different human diseases. Over 60 different types of HPV have been distinguished, based on the extent of cross hybridisation between their genomes, and of those, different subgroups are associated primarily with different types of disease. For example HPVs of types 1, 2, etc are associated with cutaneous warts of the hands and feet. HPVs 5 and 8 are associated with the rare disorder epidermodysplasia verruciformis.
Approximately twenty HPV types infect the genital mucosa, and can be divided into two subsets on the basis of severity of the disease with which they are associated. The first group include viruses such as HPV-6 and HPV-11 which are associated with the majority of benign condylomas (warts), including genital warts. The second group includes HPVs 16, 18, 31, 33 and 45, associated with flat warts of the cervix, and involved in malignant conversions leading to carcinomas of the uterine cervix (zur Hausen, Cancer Res, 49 (1989) pp 4677-4681).
Diseases associated with HPV are generally characterised by benign proliferations of epithelial tissue (warts) caused directly by virus infection. The virus infects the basal non-keratinised cells of the epithelium but cannot complete its replication cycle in these cells. Instead virus gene expression is limited to a set of early proteins which can induce the infected cell to proliferate, giving rise to the characteristic wart. In the upper layers of the wart however, the infected cells begin to undergo terminal differentiation towards their final keratinised state, and this differentiation is sufficient to allow the virus to complete its replication cycle with the production of virus proteins and ultimately new virus particles.
These lesions, although proliferative, are at low risk of malignant conversion, and in most cases the warts remain benign. However, in some cases, over a period of years, cells carrying HPV sequences may become tumorigenic. In this case it appears that the bulk of the virus genome is likely to be lost, and a residual portion of the genome, usually including the virus E6 and E7 genes becomes integrated into the genome. This progression to malignant cancer is, however, associated primarily with a limited range of HPV types, namely HPVs 16, 18, 31, 33, 35, 45 and with particular tissues such as the cervix and penis.
It is known that the immune system can play a role in controlling HPV infection. It is well known that the incidence of HPV-induced skin warts and HPV-associated diseases increases in those who are receiving immunosuppressive treatment, suggesting that in many cases virus infection is kept under control by immunological mechanisms. Further evidence for the capacity of the immune system to control infection has come from study of spontaneous wart regression. A common observation is that in some individuals with genital warts, the warts suddenly disappear. Such regressing warts have been studied histologically, revealing a substantial influx of T lymphocytes in the lesions. Regression is believed to be mediated by the immune system.
Effective immune responses to HPV infections are thought to be mainly cell-mediated since disease can persist in individuals with serum antibodies against HPV. Moreover, it is known that spontaneous regression of warts is often accompanied by lymphocytic infiltration, itching, reddening of the affected area and other symptoms characteristic of cell-mediated immune reaction. HPV infections are also common in patients with impaired cellular immunity, where persistence of viral disease suggests poor immune surveillance.
Studies on regression of papillomavirus-associated disease in vaccinated animal models also support the concept of an immune effector in combatting disease. For example, cattle vaccinated against Bovine papillomavirus (BPV) produced antibodies reported not to be neutralising, yet vaccination with a fusion protein comprising sequences of BPV L2 and non-HPV sequences (beta galactosidase) has been shown to produce both prophylactic and therapeutic responses in these animals: (see WO93/00436, of Cancer Research Campaign Technology Limited: Jarrett et al, and Jarrett et al, Virology, 184 (1991) pp 33-42).
The use of HPV proteins such as L1, L2 in the preparation of vaccines is known for example from WO 93/02184 (Univ of Queensland & CLS Ltd: I Frazer et al: Papilloma virus vaccine). Other HPV proteins have been decribed for use in immunodiagnostics, e.g. in WO 91/18294 (Medscand AB: J Dillner et al: Synthetic peptides of various human papillomaviruses, for diagnostic immunossay); and EP 0 375 555 (Medgenix: G De Martynoff et al: Peptides, antibodies against them, and methods for detection and dosage of papilloma virus).
EP 0 456 197 (1991) (Behringwerke: C Bleul et al) discloses peptides with one or more seroreactive epitopes of defined sequence from HPV18 proteins E1, E6, and E7. EP 0 451 550 (1991) (Behringwerke: M Mueller et al) discloses peptides with one or more seroreactive epitopes of defined sequence from HPV16 proteins E4, E6, E7, or L1. The disclosures are for screening test purposes, and also mention vaccine use.
WO 93/00436 (Cancer Research Campaign Technology: WFH Jarrett et al) disclose papillomavirus proteins and fragments related to L2 protein for prophylaxis and therapy of papillomavirus tumours, also mentioning preparation of E7 protein.
WO 92/16636 (Immunology Ltd: MEG Boursnell et al) discloses genetic sequences of HPV16 and HPV18 E6 and E7 as fused genes inserted in a recombinant vaccinia virus vector, causing in-vivo expression of antigens after administration of the live virus vector.
WO 92/05248 (Bristol-Myers Squibb: EK Thomas et al) proposes materials for inhibiting and treating human papillomavirus infection and cell transformation, mentioning recombinant cells (including virus vectors) containing a gene encoding a peptide substantially corresponding to a region of the E6 and/or E7 gene product or a chimeric peptide compound of one or more regions of HPV proteins.
CP Crum et al, Virology 178 (1990) pp 238-246, describe expression of fused part-sequences of HPV-16 L1 and E4 and the use of the proteins with Freund's complete adjuvant to immunise rabbits to make antisera for diagnostic or other test purposes.